Project Summary Whereas classic organelles such as the endoplasmic reticulum and mitochondria are compartments defined by lipid layer membranes, it has become apparent over the past decade that a plethora of ?membrane-less organelles/compartments?, aka ?biomolecular condensates? formed and maintained through the intracellular phase separation of biomolecules, play equally important roles in the proper function (and thus malfunction) of the cell. Here we propose to resolve, at ~10 nm spatial resolution, both the structure and the local physical properties of phase-separated systems relevant to cellular physiology. We will achieve these goals with a novel optical tool we recently developed, namely, spectrally resolved, functional super-resolution microscopy. Considering the nature of phase separation in cells, we focus on probing local hydrophobicity (chemical polarity), which often drives the phase separation of biomolecules from the aqueous cytosol, but also examine other local physical and chemical parameters, including pH and viscosity, across different phases. These experiments will help clarify what really defines a ?phase? at the nanoscale, and answer key questions on how densely the different types of biomolecular condensates are packed, how homogenous the physical properties is at the nanoscale within each separated phase and across different phases, and how the phase-separated biomolecular condensates grow and mature over time. Our work will thus help redefine how phase separation modulates the proper function of the cell.